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REVIEW ARTICLEpublished: 17 July 2013

doi: 10.3389/fphar.2013.00088

Connexin and pannexin hemichannels in brain glial cells:properties, pharmacology, and rolesChristian Giaume1,2,3*, Luc Leybaert 4, Christian C. Naus5 and Juan C. Sáez 6,7

1 Collège de France, Center for Interdisciplinary Research in Biology/Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7241/InstitutNational de la Santé et de la Recherche Médicale U1050, Paris, France

2 University Pierre et Marie Curie, Paris, France3 MEMOLIFE Laboratory of Excellence and Paris Science Lettre Research University, Paris, France4 Physiology Group, Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium5 Department of Cellular and Physiological Sciences, Life Sciences Institute, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada6 Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago, Chile7 Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Valparaíso, Chile

Edited by:

Aida Salameh, Heart CentreUniversity of Leipzig, Germany

Reviewed by:

Gregory E. Morley, New YorkUniversity School of Medicine, USARoger J. Thompson, University ofCalgary, CanadaPanagiotis Bargiotas, University ofHeidelberg, Germany

*Correspondence:

Christian Giaume, Collège de France,Center for Interdisciplinary Researchin Biology/Centre National de laRecherche Scientifique, Unité Mixtede Recherche 7241/Institut Nationalde la Santé et de la RechercheMédicale U1050, 11 Place MarcelinBerthelot, 75231 Paris Cedex 05,Francee-mail: [email protected]

Functional interaction between neurons and glia is an exciting field that has expandedtremendously during the past decade. Such partnership has multiple impacts on neuronalactivity and survival. Indeed, numerous findings indicate that glial cells interact tightly withneurons in physiological as well as pathological situations. One typical feature of glial cellsis their high expression level of gap junction protein subunits, named connexins (Cxs), thusthe membrane channels they form may contribute to neuroglial interaction that impactsneuronal activity and survival. While the participation of gap junction channels in neuroglialinteractions has been regularly reviewed in the past, the other channel function of Cxs, i.e.,hemichannels located at the cell surface, has only recently received attention. Gap junctionchannels provide the basis for a unique direct cell-to-cell communication, whereas Cxhemichannels allow the exchange of ions and signaling molecules between the cytoplasmand the extracellular medium, thus supporting autocrine and paracrine communicationthrough a process referred to as “gliotransmission,” as well as uptake and release ofmetabolites. More recently, another family of proteins, termed pannexins (Panxs), has beenidentified. These proteins share similar membrane topology but no sequence homologywith Cxs. They form multimeric membrane channels with pharmacology somewhatoverlapping with that of Cx hemichannels. Such duality has led to several controversiesin the literature concerning the identification of the molecular channel constituents (Cxsversus Panxs) in glia. In the present review, we update and discuss the knowledge of Cxhemichannels and Panx channels in glia, their properties and pharmacology, as well as theunderstanding of their contribution to neuroglial interactions in brain health and disease.

Keywords: astrocytes, oligodendrocytes, microglia, gap junctions, neuroglial interactions

INTRODUCTIONFor a long time, it has been taken as dogma that connexin (Cx) pro-teins can only function as gap junction channels. Indeed, before theaggregation of Cxs at the junctional plaque and subsequent forma-tion of gap junctions, hexameric rings of Cxs, termed connexons,were initially assumed to remain closed. An obvious reason for thisocclusion was that, as gap junction channels are “poorly” selectivefor ions and permeable to low molecular weight molecules (<1to 1.2 KDa), if once at the membrane connexons could open,the cell would lose its integrity or at least would have to spendsubstantial energy to maintain this energetically unfavorable con-dition. Such statements began to be challenged when evidencefor “functional hemichannels,” a term proposed to substitute forplasma membrane connexons, were reported in the early 1990s.In these pioneering studies, Cx hemichannels were opened eitherby large depolarization in Xenopus oocytes (Paul et al., 1991) orby lowering the extracellular calcium ion (Ca2+) concentrationin horizontal cells (DeVries and Schwartz, 1992). Later on, the

occurrence of functional hemichannels (i.e., hemichannels thatcan be turned into the open state) composed of Cx43 was demon-strated in primary cultures of astrocytes in the absence of externalCa2+ (Hofer and Dermietzel, 1998). This observation was fol-lowed by the demonstration that metabolic inhibition performedin the presence of normal external Ca2+ concentrations (1–2 mM)induced cell permeabilization, due to Cx43 hemichannel open-ing, before loss of membrane integrity (Contreras et al., 2002).More recently, hemichannel opening in astrocytes was triggeredeither by treatment with pro-inflammatory cytokines or selec-tive lipopolysaccharide (LPS) stimulation of microglia co-culturedwith astrocytes, again in the presence of external Ca2+ (Retamalet al., 2007a). The opening of Cx43 hemichannels in astrocyteswas also demonstrated to occur in experiments designed to deci-pher the mechanism of intercellular Ca2+ wave propagation inastrocytes to which both gap junction channels and hemichannelscontribute (Scemes and Giaume, 2006; Leybaert and Sanderson,2012). In this case, the release of ATP through Cx43 hemichannels

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Giaume et al. Hemichannels in brain glial cells

turned out to play a key role in Ca2+ wave propagation mediatedby an extracellular paracrine signaling component (Cotrina et al.,1998; Arcuino et al., 2002). All these initial experiments on Cxhemichannels utilized astrocytes as a cell model giving to thesecells new roles in neuroglial interaction (Bennett et al., 2003).

At the turn of the century another membrane channel fam-ily, the pannexins (Panxs), was identified (Panchin et al., 2000)and demonstrated to be orthologs of innexins, the gap junctionproteins expressed in invertebrates (Baranova et al., 2004). So far,there are no reports of gap junctional communication supportedby native Panxs, although over expression of Panx1 enhances gapjunctional coupling in glioma cells (Lai et al., 2007). They formmembrane channels contributing to membrane permeabilization,similar to Cx hemichannels, and they are expressed in glial cells(Orellana et al., 2009; Scemes, 2012). The aim of the present reviewis to summarize what is actually known about Cx hemichannelsand Panx channels in glia (mainly astrocytes and microglia) and todiscuss their contribution to neuroglial interactions taken in thenormal and pathological context. An overview of methods andapproaches to investigate Cx hemichannels and Panx channels hasrecently been published (Giaume et al., 2012); we refer the readerto this paper for a more detailed discussion of methodologicalaspects.

CONNEXIN AND PANNEXIN EXPRESSION IN GLIAL CELLSGap junctions provide a unique direct conduit between cells,influencing crucial cellular processes through activities includ-ing electrical conductance and metabolic cooperation, mediatedby the passage of ions, amino acids, glucose, glutathione, ATP,and small signaling molecules (reviewed in Sohl and Willecke,2004), as well as microRNAs (Katakowski et al., 2010). When oneconsiders Cx expression in the brain (Theis et al., 2005), it is evi-dent that they are most abundant in glia, namely the astrocytes,oligodendrocytes, and microglia (Rouach et al., 2002). With thegenomic characterization of Cx (Willecke et al., 2002) and Panx(Baranova et al., 2004) gene families, it has been determined thatthere are at least 10 Cxs and 2 Panxs expressed in the brain (seebelow). There is also a temporal (i.e., developmental) and spatial(i.e., cell type and brain region) pattern regarding this expressionand localization. Furthermore, these channel proteins can havedifferent roles in different cell types, either as the canonical gapjunction intercellular channel or as a hemichannel (Goodenoughand Paul, 2003; Figure 1). Cx hemichannels and Panx1 channelsmediate the release of ATP that activates P2 receptors, promotingrises in intracellular Ca2+ concentrations; gap junction channelsmediate the intercellular transfer of second messenger propa-gated calcium signal (Suadicani et al., 2004; Anselmi et al., 2008;Leybaert and Sanderson, 2012). Under pathological conditions,Panx1 channels have been proposed to mediate activation of theinflammasome both in neurons and astrocytes (Iglesias et al., 2009;Silverman et al., 2009), whereas Cx43 hemichannels are involvedin acceleration of astroglia and neuronal cell death triggered byhypoxia-reoxygenation in high glucose (Orellana et al., 2010) andβ-amyloid peptide, respectively (Orellana et al., 2011b,c). A furtherlevel of complexity can be envisioned in the context of Cxs, wherethe heteromeric assembly of different Cx proteins into a singlechannel can result in distinct differences in biophysical properties

(Bevans et al., 1998; Harris, 2007). This feature is also true forPanxs since Panx1 can form both homomeric and heteromericchannels with Panx2 (Bruzzone et al., 2005). Thus for specific cel-lular functions, the complement of Cxs and Panxs expressed isimportant in the physiological or pathological context being con-sidered. While other “non-channel” functions are emerging forCxs (Vinken et al., 2012) and Panxs (MacVicar and Thompson,2010), this review will be limited to channel-related functions andproperties.

While this review will focus on gap junction channels andhemichannels in brain glial cells, it is important to note thatgap junction proteins have also been identified in brain neurons(Andrew et al., 1981; Söhl et al., 2005). These include Cx26, Cx30.2,Cx36, Cx45, and Cx57 (Rouach et al., 2002). Neurons also expressPanx1 in vivo (Bruzzone et al., 2003), and recently it has beenreported that neural progenitors in the adult rodent hippocampusexpress Panx2 (Swayne et al., 2010). It is thus possible, and has beenreported in some situations, that neurons form gap junctions withastrocytes since some of the same Cxs are expressed in both celltypes (Fróes et al., 1999; Alvarez-Maubecin et al., 2000). Astro-cytes express multiple Cxs, including Cx43, Cx26, Cx30, Cx40,Cx45, and Cx46 (Giaume et al., 2012), as well as Panx1 (Igle-sias et al., 2009) and Panx2 (Zappala et al., 2007). Non-treatedmicroglia express Cx43 (Eugenín et al., 2001; Garg et al., 2005),Cx36 (Dobrenis et al., 2005), and Cx32 (Maezawa and Jin, 2010),as well as Panx1 (Orellana et al., 2011b). Finally, oligodendro-cytes express Cx32, Cx47, and Cx29 in vivo (Altevogt et al., 2002;Wasseff and Scherer, 2011). The complexity of Cxs and Panxsfunctioning as gap junction channels/hemichannels and channels,respectively, in these various cell types can be viewed as providing abackground level of activity to support and modulate the complexneural activity mediated via gliotransmission.

The expression profiles noted have been associated with matureneurons and glial cell types. However, these various cell types arisedevelopmentally from neuronal and glial precursors. Several stud-ies have demonstrated changes in expression of Cxs and Panxsduring brain development (reviewed in Vogt et al., 2005; Belousovand Fontes, 2013). Given that several reports have describeddisturbed neural development when expression of some Cxs ismodified through the use of gene knockdown or knockout strate-gies, gap junction channels and hemichannels are likely to playsignificant roles in processes including cell proliferation, migra-tion, and differentiation (Fushiki et al., 2003; Elias et al., 2007;Wiencken-Barger et al., 2007; Cina et al., 2009; Liu et al., 2012).

Initial studies on Cx functions in neural cells in culture and inbrain slices focused on their expected role in forming gap junctionchannels, confirming their function via electrophysiological anal-ysis or methods using low molecular weight dye tracers, which canpass from cell to cell through these intercellular channels (Giaumeet al., 1991). Therefore, many of the phenotypic changes observedwhen enhancing or decreasing Cx expression were attributed tochanges in gap junctional communication. With the recognitionthat Cxs can also form hemichannels, the consequences of Cxexpression began to be considered in a new way (Contreras et al.,2004), and appropriate methods developed to assess their presenceand activity (Giaume and Theis, 2010). Cx hemichannels and Panxchannels are more typical of most membrane channels, like ion

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FIGURE 1 | Schematic showing that connexins can from both hemichannels and gap junction channels. In contrast, pannexins normally form channelsequivalent to hemichannels. See text for details (provided by Stephen Bond, Ph.D., UBC).

channels, in their direct communication between intracellular andextracellular space. However, they are unique in their pore size andconductance properties (Thompson and Macvicar, 2008).

The formation of gap junction channels and hemichannelsby Cxs can help explain some contradictory effects of alteringCx expression. For example, enhanced Cx43 expression in brainglial cells, in culture and in vivo, has been shown to be bothneuroprotective and neurodestructive in ischemic and excitotoxicsituations (reviewed in Orellana et al., 2009; Kozoric and Naus,2013). Recent studies have indicated that gap junction channelsformed by Cx43 can be neuroprotective, while Cx43 hemichan-nels may be more detrimental to neuronal survival. The followingsections of this review provide further insight into the distinctproperties of hemichannels and how these impact the progressionof neurological disorders.

Cx HEMICHANNEL AND Panx CHANNEL PROPERTIES IN GLIABIOPHYSICSToday, most Cxs expressed in rodent glial cells, including Cx26,Cx30, Cx32, Cx43, and Cx45, have been shown to formfunctional hemichannels in endogenous and/or exogenous expres-sion systems (Bennett et al., 2003). Nevertheless, it remainsunknown if this is also the case for Cx29 and Cx47 found in

oligodendrocytes. In exogenous expression systems, Cx30, Cx32,Cx43, and Cx45 hemichannels show a very low open probabil-ity at resting membrane potential and normal concentration ofthe extracellular divalent cations, Ca2+ and Mg2+. For Cx26hemichannels, these properties are species-specific; human andsheep but not rat Cx26 forms hemichannels that at resting mem-brane potential and normal concentration of extracellular divalentcations show opening activity but the rat protein is characterizedby a single evolutionary amino acid change (D159N) that limitsthis feature (González et al., 2006).

Opening of Cx hemichannels studied so far can be promotedby positive transmembrane voltages and reduced concentrationsof extracellular divalent cations (Bennett et al., 2003). Theseconditions have been most frequently used to gain knowledgeon biophysical features of hemichannels, including permeabilityproperties (see below), identification of voltage sensitivity, unitaryconductance, kinetic properties, and polarity of closure. However,other gating mechanisms can also promote opening of hemichan-nels, as described below (see Regulation). In general, hemichannelsshow a time- and voltage-dependent macroscopic current, activat-ing with depolarization and inactivating with hyperpolarization.However, this feature is not equal in hemichannels formed bydifferent Cxs. For example, the voltage dependency is strong




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for mouse Cx30 hemichannels (Valiunas and Weingart, 2000)but detectable only at very high positive potentials for rat Cx43hemichannels (Contreras et al., 2003). For Cx32 hemichannels,the conductance is determined by charges dispersed over the porepathway (Oh et al., 2008). Hemichannels formed by human Cx26or rat Cx43 show bipolar voltage behavior (Contreras et al., 2003;González et al., 2006); however, this appears to be less well estab-lished for Cx43, for which unitary current events were foundto monotonically rise in the voltage range of +30 to +90 mV(Wang et al., 2012). Hemichannels present two distinct voltage-gating mechanisms, which are called loop- (slow) and V(j)- (fast)gating, respectively (Contreras et al., 2003; Oh et al., 2008). ForCx32 hemichannels the loop-gate is mediated by a conformationalchange that reduces the pore diameter in a region located closeto the first transmembrane domain and first extracellular loop(Bargiello et al., 2012). It was recently also proposed that the intra-cellular pore entrance narrows from 15 to 10 Å with loop-gatedbut not with V (j)-gated channel closure. Moreover, it was shownthat the extracellular entrance does not undergo evident largeconformational changes with either voltage-gating mechanisms(Kwon et al., 2013). The unitary conductance is characteristic forhemichannels with specific Cx composition; the fully open state forCx26 hemichannels is characterized by ∼320 pS unitary conduc-tance (González et al., 2006), for Cx32 hemichannels this is ∼90 pS,for Cx43 hemichannels ∼220 pS (Contreras et al., 2003; Reta-mal et al., 2007a; Wang et al., 2012) and for Cx45 hemichannels∼57 pS (Valiunas, 2002). They also present conductance substatesas a result of an inactivation mechanism that hinders completeactivation of hemichannels under prolonged depolarization (Vali-unas, 2002; Contreras et al., 2003; Gómez-Hernández et al., 2003).The most general features mentioned above have been useful todemonstrate and understand the involvement of hemichannelsin different physiologic functions and also to identify hemichan-nels with specific Cx composition in glial cells (Orellana et al.,2011c). However, much more remains to be investigated since Cxsare post-translationally modified and thus, their biophysical fea-tures might be affected according to the metabolic state of thecell.

The biophysical properties of Panx channels are much less doc-umented than those of Cx hemichannels, possibly as a result oftheir more recent discovery. Panx1 channels have been so fardemonstrated to be present in microglia, astrocytes, and oligo-dendrocytes (Iglesias et al., 2009; Domercq et al., 2010; Orellanaet al., 2011a), whereas Panx2 is expressed in post-ischemic astro-cytes (Zappala et al., 2007). The open state of Panx1 channels ispromoted by positive membrane voltages and elevation of intra-cellular free Ca2+ concentration. Voltage clamp experiments haveindicated widely divergent unitary conductances for Panx1 chan-nels, varying from 68 to 550 pS (Locovei et al., 2006; Kienitz et al.,2011; Ma et al., 2012). This controversy in the single channel con-ductance of Panx1 channels has not been resolved yet and mightbe explained in part by differences in recording conditions or solu-tions, cell type, phenotypic differences, and post-translationalmodifications. It could be partially explained by simultaneousopening of channels and thus, high conductance values mightrepresent multiples of a single channel with a lower conductancevalue. An issue to keep in mind is that biophysical properties

of exogenously expressed Panx channels might be different fromthose expressed simultaneously with all membrane and intracel-lular proteins (e.g., P2X receptors) that might interact with themin endogenous expression systems.

PERMEABILITYIn addition to selective ion channels or transporters, other chan-nels can alter the permeability of the cell membrane underphysiological and pathological conditions (Schalper et al., 2009).The origin of these permeability changes could be Cx- andPanx-based channels located at the cell surface. However, othermembrane channels might also be involved in drastic changes inmembrane permeability [e.g., P2X7 receptors, transient receptorpotential (TRP) channels and calcium homeostasis modulator 1(CALHM1) ion channel] and their possible involvement shouldbe proved or disproved when testing the permeability of Cxhemichannels or Panx channels in a particular cell type.

Net changes in membrane permeability due to hemichannelshave been most frequently studied through the use of inhibitors ormolecular biology approaches that induce or abolish the expres-sion of Cxs or Panxs. With these experimental approaches it hasbeen proposed that hemichannels (Panx1 and/or Cx26, Cx32,and Cx43) allow the release of precursor or signaling molecules[e.g., including NAD+, ATP, adenosine, inositol trisphosphate(IP3), and glutamate; Bevans et al., 1998; Stout et al., 2002;Ye et al., 2003; Anderson et al., 2004; Bao et al., 2004a; Locoveiet al., 2006; Gossman and Zhao, 2008; Kang et al., 2008; Songet al., 2011], uptake of second messengers [e.g., cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate(NAADP), nitric oxide (NO), and Ca2+; Heidemann et al., 2005;Sánchez et al., 2009, 2010; Schalper et al., 2010; Song et al., 2011;De Bock et al., 2012; Figueroa et al., 2013) and uptake of variousmetabolites (e.g., glucose, glutathione, and ascorbate; Bruzzoneet al., 2001; Ahmad and Evans, 2002; Rana and Dringen, 2007;Retamal et al., 2007a; see Table 1 for Cx43 hemichannels andPanx1 channels). In addition, it has been demonstrated that Cxhemichannels are permeable to synthetic molecules [e.g., Luciferyellow, ethidium, calcein, propidium, 5(6)-carboxyfluorescein,and 2-(4-amidinophenyl)-1H-indole-6-carboxamide (DAPI); forreview, see Sáez et al., 2010]. The fact that Cx26 and Cx32hemichannels reconstituted in lipid membranes are permeable toCa2+ and cyclic nucleotides, respectively (Harris, 2007; Schalperet al., 2010; Fiori et al., 2012), indicates that no additional bindingpartners or associated molecules are required for hemichannels tofunction as conduits for small molecules or ions across the cellmembrane. Since the membrane permeability change due to tran-sient hemichannel opening may allow release of enough moleculesto reach effective concentrations in a confined extracellular space,astroglial hemichannels have been proposed as membrane path-ways for paracrine/autocrine cell signaling with physiological andpathological implications for glial and neuronal cells (Orellanaet al., 2011c; Stehberg et al., 2012).

Membrane permeability changes via Cx hemichannels are inpart explained by variations in the number of hemichannelsavailable at the cell surface. With regard to this, an increase inthe number of surface Cx43 or Cx45 hemichannels explains theincrease in membrane permeability induced by fibroblast growth




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Table 1 | Overview of substances passing through glial Cx hemichannels and Panx channels and compounds that block these membrane

pathways.

Models Techniques Permeant metabolic or

signaling molecules

Hemichannel blocking

condition used

Reference

Astrocyte cultures Dye uptake 2-NBDG (uptake) Cx43, lanthanum (La3+),

Gap27

Retamal et al. (2007a)

Astrocyte cultures Colorimetric Tietze

method

Glutathione (release) Divalents, alcohols, flufenamic

acid, carbenoxolone

Rana and Dringen (2007)

Astrocyte cultures HPLC analysis Glutamate, aspartate

(release)

Divalents, alcohols, flufenamic

acid, carbenoxolone

Ye et al. (2003)

Acute hippocampal slices,

C6 transfected cells

Bioluminescence ATP

imaging

ATP (release) Cx43, non-transfected C6 cells Cotrina et al. (1998);

Kang et al. (2008)

Astrocyte cultures Luciferin/luciferase assay ATP (release) Panx1, Panx1 knock out

mouse

Suadicani et al. (2012)

Microglia cultures treated

with amyloidβ

Luciferin/luciferase

bioluminescence and

enzyme-linked

fluorimetric assays

ATP, glutamate (release) Panx1 and Cx43, 10Panx1,

probenecid

Orellana et al. (2011b)

Microglia cultures treated

with TNF-α

Colorimetric assay Glutamate (release) Cx32, Gap24, carbenoxolone Takeuchi et al. (2006)

This table summarizes most if not all data available on signaling molecules passing through Cx or Panx channels (in bold, column 4) and conditions or blockers of theCx hemichannels or Panx channels used in each particular study. Citations refer to the initial demonstration of the channel permeation property.The model, method ofdetection of the permeant compound, and reference where these findings were described are also included. 2-NBDG [2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-deoxyglucose] is a fluorescent glucose analog.

factor-1 (FGF-1) in cells that express these proteins (Schalper et al.,2008b). However, in other conditions such as exposure to extra-cellular solution without divalent cations (Schalper et al., 2008a)or tanycytes treated with extracellular glucose (Orellana et al.,2012a), the increase in membrane permeability is explained byopening of hemichannels via a gating mechanism. In both casesthere is a net increase in membrane permeability mediated byhemichannels, possibly without changes in intrinsic permeabilityproperties of hemichannels themselves. Nonetheless, changes inmembrane permeability could also result from alterations in per-meability features of hemichannels. For instance, protein kinaseC (PKC)-mediated phosphorylation of the six subunits of Cx43hemichannels reconstituted in lipid membranes reduces the porepermeability allowing permeation of the small hydrophilic soluteethylene glycol (Mr 62) but not sucrose (Mr 342; Bao et al.,2007). Changes in other hemichannel permeability properties,such as affinity or charge selectivity induced by covalent mod-ification including phosphorylation by other protein kinases ornitrosylation, cannot be ruled out yet.

The permeability properties depend on the size, shape, andnet charge of each permeant ion or molecule while the concen-tration gradient across the cell membrane acts as the drivingforce. Since the study of permeability characteristics of a mem-brane transporter should be done with a rapid time scale (withina few seconds to a minute) it is important to have a library ofpermeant molecules that allow real time determination of theirdisappearance or appearance. Transport via Cx43 hemichannels

has been shown to be cooperative and saturable with parameters(e.g., V max, Km, and Hill coefficient) depending on the perme-ant cationic species (Orellana et al., 2011a). These findings implythat opening of hemichannels does not necessarily lead to loss ofimportant intracellular molecules because of competition effectswith other, less important molecules or ions and this interpreta-tion might be valid to transport in both directions across the cellmembrane. Of note, the permeability properties of hemichannelsto anionic molecules have not been reported yet. To the best of ourknowledge, the permeability properties of Panx1 channels remainequally unknown. It is also not known whether channels formedby different Cxs or Panxs show different permeability propertiessuch as size exclusion and charge selectivity. In general, quantita-tive kinetic properties of permeation of Cx hemichannels and Panxchannels remain largely unexplored. Table 1 summarizes currentevidence on the permeability of Cx43, Cx32 hemichannels, andPanx1 channels in brain glial cells.

REGULATIONHemichannels are regulated by diverse conditions of the extra-cellular and intracellular microenvironments. They include Ca2+concentration outside and inside the cell, monovalent cation con-centration, pH, mechanical stress, extracellular ligands, proteinkinases, protein phosphatases, as well as oxidant and reducingagents. Here, the most recent studies not included in previousreviews (Sáez et al., 2010; Wang et al., 2013), will be pres-ented.




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Hemichannel opening and modulation by Ca2+Unapposed Cx hemichannels of cells in culture are preferentiallyclosed but can be opened by several trigger conditions (reviewedin Sáez et al., 2005, 2010). These include, transmembrane voltagewith activation threshold of −30 mV for Cx30 and +50 mV forCx43 in the presence of normal extracellular Ca2+ (Valiunas andWeingart, 2000; Wang et al., 2012), mechanical forces/strain (Baoet al., 2004b; Luckprom et al., 2011), a decrease of extracellularCa2+ (Li et al., 1996; Stout et al., 2002; Ye et al., 2003; Ramachan-dran et al., 2007; Torres et al., 2012), an increase of intracellular freeCa2+ (De Vuyst et al., 2006, 2009; Wang et al., 2012), alterations inphosphorylation status (Bao et al., 2004c) and redox status (Reta-mal et al., 2007b), and ischemia-mimicking conditions (John et al.,1999; Contreras et al., 2002; Wang et al., 2013).

Role of extracellular Ca2+. A decrease of extracellular divalentcation concentration was one of the first conditions reportedto stimulate Cx43 hemichannel opening, as introduced above.Work in a hepatoma cell line expressing Cx43 demonstrated thata decrease of the extracellular Ca2+ concentration triggered theuptake of Lucifer yellow, a hemichannel-permeable fluorescentdye (Li et al., 1996). Similar observations were obtained in astro-cytes, where hemichannel opening was demonstrated to triggerthe release of glutamate and aspartate (Ye et al., 2003). Lower-ing extracellular Mg2+ appeared to promote low Ca2+-triggeredhemichannel opening but Ca2+ appeared to be the dominant fac-tor. It is thought that normal physiological extracellular Ca2+concentrations (∼1.8 mM) keep non-junctional Cx hemichannelsclosed in the plasma membrane, until they become incorporatedinto gap junction channels where they open by a process called“loop gating” as a consequence of extracellular loop interactions.In general, the half-maximal effective concentration for Ca2+-related opening of Cx43 hemichannels is in the order of 100 μMextracellular Ca2+. The proposed mechanism of millimolar Ca2+closure of Cx hemichannels involves an interaction of extracel-lular Ca2+ with a Ca2+ binding site that consists of a ring ofaspartate residues at the extracellular vestibule of the hemichan-nel pore thereby closing the channel (Gómez-Hernández et al.,2003). Such a mechanism has been best documented for Cx32 butappears to be also plausible for the astroglial Cxs, Cx30 and Cx43.Recently, Torres et al. (2012) have elegantly exploited the extracel-lular Ca2+-sensitivity of astroglial hemichannels to trigger theiropening by photo-activating a Ca2+ buffer (thereby increasing itsCa2+ affinity) added to the interstitial fluid of a hippocampal acuteslice preparation (discussed further below under Section “Impactof Hemichannel-Mediated Gliotransmission on Synaptic Activityand Behavior”).

Role of intracellular Ca2+. Besides extracellular Ca2+, intracel-lular cytoplasmic Ca2+ also influences hemichannel function. Infact, it was found that Cx hemichannel opening triggered by adecrease of extracellular Ca2+ could be inhibited by ester-loadingthe cells with the Ca2+ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), added to dampen intracel-lular Ca2+ changes (De Vuyst et al., 2006). Further investigationsdemonstrated that Cx32 hemichannels are activated by moder-ate (below 500 nM) changes in intracellular Ca2+ and inhibited

by Ca2+ changes above 500 nM. Subsequent work showed thatCx43 hemichannels are also characterized by such a biphasic, bell-shaped, response to changes in intracellular Ca2+ concentration,in ATP release hemichannel studies as well as in dye uptake studiesin glioma cells and other cell types (De Vuyst et al., 2009; De Bocket al., 2012). Recently, single channel studies of Cx43 hemichan-nels robustly confirmed these observations (Wang et al., 2012).Cx hemichannels are Ca2+ permeable (Sánchez et al., 2009, 2010;Schalper et al., 2010; Fiori et al., 2012) and the biphasic depen-dency on intracellular Ca2+ thus confers a positive feedback below500 nM intracellular Ca2+ concentration (Ca2+-induced Ca2+entry via Cx43 hemichannels) and negative feedback at higherconcentrations. Interestingly, IP3 receptor channels present inthe endoplasmic reticulum (ER) are Ca2+ release channels thathave a similar bell-shaped Ca2+ dependency (Bezprozvanny et al.,1991). Positive and negative Ca2+ feedback at IP3 receptor chan-nels forms the basis of Ca2+ oscillations, thus, it was hypothesizedthat Ca2+ feedback at Cx hemichannels could also play a role inCa2+ oscillations (De Bock et al., 2012). Experimental work withvarious peptides targeting Cx hemichannels indeed demonstratedthat inhibiting hemichannel opening, or preventing their closurein response to high intracellular Ca2+, blocked Ca2+ oscillationstriggered by bradykinin. Thus, Cx hemichannels constitute a puta-tive gliotransmitter release pathway. Also, they may contributeto Ca2+ oscillations in astrocytes and thereby potentially lead togliotransmitter release via other Ca2+-dependent pathways (Olietand Mothet, 2006; Zorec et al., 2012). In brain endothelial cells,it was found that Cx hemichannel blocking peptides preventedthe opening of the blood–brain barrier, triggered by bradykinin,by inhibiting Ca2+ oscillations in the endothelial cells (De Bocket al., 2011). Below, we discuss in more detail some peptide-basedhemichannel inhibitors.

Influence of monovalent cationsElevations in extracellular K+ shift the activation potentialof Panx1 hemichannels to a more physiological range. Panx1hemichannels expressed in astrocytes might serve as K+ sensorsfor changes in the extracellular milieu such as those occurringunder pathological conditions (Suadicani et al., 2012). This type ofregulation might also occur under physiological conditions withhigh neuronal activity. Thus, elevations in extracellular K+ ofthe magnitude occurring during periods of high neuronal activ-ity have been proposed to affect the intercellular signaling amongastrocytes (Scemes and Spray, 2012). Accordingly, the gliotrans-mitter release in the hippocampus in response to high neuronalactivity is sensitive to P2 receptor and Panx1 channel blockers(Heinrich et al., 2012). Cx30 gap junction channels have recentlybeen demonstrated to be regulated by the concentration of exter-nal K+ (Roux et al., 2011); whether astroglial Cx hemichannels areinfluenced by the extracellular K+ concentration is currently notknown.

Influence of pHUnder pathological conditions, the intracellular and extracellu-lar pH may display abrupt changes. Related to this issue and inthe presence of extracellular divalent cations, Cx43 hemichannelsare opened by alkaline pH applied at the extracellular side and




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preferentially closed at physiologic pH (Schalper et al., 2010).The activity of Cx45 hemichannels recorded at positive mem-brane potentials and in the absence of extracellular Ca2+ isdrastically reduced by acidification (Valiunas, 2002). In addi-tion, the pH sensitivity might be potentiated by protonatedaminosulfonates (Bevans and Harris, 1999), such as taurine thatcould be in millimolar concentration in the cytoplasm of sev-eral cell types including astrocytes (Olson, 1999). This effectis Cx specific; Cx26 but not Cx32 hemichannels are closed byaminosulfonates at constant pH (Bevans and Harris, 1999). At theultrastructural level (high-resolution atomic force microscopy),Cx26 hemichannels are closed at pH < 6.5 [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer] and openedreversibly by increasing the pH to 7.6 (Yu et al., 2007). More-over, molecular studies have revealed that Cx26 hemichannelclosure induced by taurine requires the interaction between thecytoplasmic loop and the C-terminal (CT; Locke et al., 2011).

Influence of mechanical forcesMechanical stress is another condition that might be relevantunder pathological situations. For example, after brain trauma theextracellular medium becomes hypertonic due to the release ofintracellular solutes to the extracellular microenvironment (Som-jen, 2002). In astrocytes, hypertonicity induces glutamate releasethrough Cx43 hemichannels (Jiang et al., 2011). This responsecould be mediated by integrin α5β1 (Batra et al., 2012), a RhoAGTPase and the contractile system (Ponsaerts et al., 2010). Thissensitivity is likely to result from the interaction between nega-tively charged amino acid residues of the CT end (Asp278 andAsp279) and a domain of the intracellular loop (D’Hondt et al.,2013). Activation of Panx1 channels by mechanical stress is notpresent in all cell types (Bao et al., 2004a; Reyes et al., 2009), sug-gesting that it should be tested in glial cells in order to validate itspossible relevance in these cells.

Influence of extracellular factors and signalsThe functional state of glial Cx hemichannels and Panx1 channelsis actively regulated by extracellular signals. In primary culturesof mouse astrocytes, stomatin inhibits Panx1-mediated whole cellcurrents by interacting with its CT (Zhan et al., 2012). In con-trast, pro-inflammatory agents such as the β-amyloid peptideincrease the surface expression and activity of Cx43 hemichan-nels and Panx1 channels in microglia and Cx43 hemichannels inastrocytes (Orellana et al., 2011c). Moreover, in brain astrocytes,epidermal growth factor (EGF) and FGF-2 inhibit Cx hemichan-nel activity via the mitogen-activated protein kinase cascade, andthe effect of the growth factors is reversed by interleukin-1β (IL-1β; Morita et al., 2007). In contrast, pro-inflammatory cytokines[tumor necrosis factor alpha (TNF-α) and IL-1β] released byLPS-activated microglia increase the activity of astroglial Cx43hemichannels via p38 kinase (Retamal et al., 2007b; Orellana et al.,2011c) and this response is inhibited by activation of CB1 recep-tors by synthetic cannabinoid agonists (Froger et al., 2009). Theorchestrated involvement of Cx hemichannels and Panx1 channelshas also been found in other experimental preparations. In spinalcord astrocytes, FGF-1 leads to vesicular ATP release followedby sequential activation of Panx1 and Cx43 hemichannels (Garré

et al., 2010). Moreover, up-regulation of astroglial Panx1 channelsand Cx43 hemichannels has been found in brain abscess (Karpuket al., 2011).

Cx43 hemichannels expressed by tanycytes, glial cells presentin the hypothalamus, open in seconds after exposure to physio-logical concentrations of extracellular glucose. This response ismediated by the sequential activation of glucosensing proteins(Orellana et al., 2012a), a response that is absent in cortical astro-cytes (Orellana et al., 2010). However, high glucose levels enhancethe increase in astroglial Cx43 hemichannel activity induced byhypoxia-reoxygenation (Orellana et al., 2010).

As a result of cell interaction with extracellular signals orconditions that affect the energy supply, intracellular metabolicpathways involving levels of free Ca2+ concentration, activityof protein kinases and phosphatases, as well as levels or activityof intracellular redox molecules, are affected. Therefore, drasticreduction in ATP levels are expected to favor the dephosphory-lated state of Cx43 hemichannels and thus, they could present anincreased activity (Bao et al., 2007). Moreover, the lack of energyleads to a rise in intracellular free Ca2+ that favors the generationof reactive oxygen and nitrogen species including NO. The latterinduce rapid nitrosylation of Cx43 and opening of hemichan-nels (Retamal et al., 2007b; Figueroa et al., 2013). This mechanismmight partially explain the increase in Cx43 hemichannel activ-ity observed in astrocytes under ischemia-like conditions sincefree radical scavengers, dithiothreitol, a sulfhydryl (SH) reducingreagent, and high intracellular levels of reduced glutathione revertthe hemichannel activity to levels found in normal cells (Contr-eras et al., 2002; Retamal et al., 2007b; Orellana et al., 2010). Finally,extracellular ATP mediates the ischemic damage to oligodendro-cytes and is partially explained by the activation of Panx1 channels(Domercq et al., 2010). In contrast to Cx43 hemichannels, S-nitrosylation of Panx1 channels reduces their activity (Lohmanet al., 2012), suggesting that their opening might be relevant onlyunder conditions where NO is overcome by −SH reducing agentssuch as reduced glutathione.

HEMICHANNEL BLOCKERS – MIMETIC PEPTIDES ANDOTHERSSpecifically targeting unapposed hemichannels is difficult becausethey are composed of the same Cx building blocks as gap junc-tion channels. As a consequence, most gap junction blockers alsoinhibit hemichannels (see Giaume and Theis, 2010). Moreover,many Cx hemichannel/gap junction channel blockers also blockPanx channels. Gap junction blockers include glycyrrhetinic acidand its derivative carbenoxolone, long-chain alcohols like hep-tanol or octanol, halogenated general volatile anesthetics likehalothane, fatty acids like arachidonic acid and oleic acid, fattyacid amides like oleamide and anandamide, and fenamates likeflufenamic acid, niflumic acid, or meclofenamic acid (reviewed inBodendiek and Raman, 2010). Most of these substances have beenused in the past to block hemichannels but the results obtainedcan only be unequivocally interpreted in terms of hemichannelswhen the contribution of gap junction channels is negligible orabsent. Substances like the trivalent ions gadolinium (Gd3+) andlanthanum (La3+) block hemichannels without inhibiting gapjunction channels or Panx1 channels, but these ions also inhibit




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other channels including maxi-anion channels (Liu et al., 2006)and Ca2+ channels (Mlinar and Enyeart, 1993). A possible way todifferentiate between Cx hemichannels and Panx channels is theuse of long-chain alcohols that block Cx hemichannels but haveonly a very small effect of Panx channels. In contrast, low concen-tration (5–10 μM) of carbenoxolone blocks Panx1 channels andonly has a minor inhibitory effect on Cx hemichannels (Schalperet al., 2008a). However, all these compounds are not specific andin addition to their inhibitory effects on Cx and Panx channels(including gap junctions channels and hemichannels) they alsoaffect other neuronal and glial membrane properties which limitstheir use (see Giaume and Theis, 2010).

A more specific targeting of Cx channels is to be expected, atleast in principle, from mimetic peptides of Cx proteins. Peptidesidentical to certain sequences on the Cx protein have been exten-sively used to interfere with the Cx channel function (Figure 2).The first Cx mimetic peptides that were introduced in the 1990swere identical to specific domains on the extracellular loops (firstor second extracellular loop) of the Cx protein. The sequencesmimicked were located in domains thought to be involved in thedocking of two hemichannels during formation of a full gap junc-tion channel (Warner et al., 1995). Exogenous addition of peptidesmimicking parts of these domains were hypothesized to interactwith yet unknown extracellular loop domains, thereby preventingextracellular loop interactions of apposed hemichannels duringdocking and thus hindering gap junction channel formation.

FIGURE 2 | Peptide tools to interfere with Cx hemichannel function.

Gap26 and Gap27 are composed of sequences located on the extracellularloops 1 (EL1) and 2 (EL2), respectively (amino acids 64–76 and 201–210,respectively) of Cx43 (mouse). These EL-mimetic peptides first inhibithemichannels and with some delay also gap junction channels. Thesequences they mimic are well conserved between different Cxs andpeptides based on Cx43 sequences could also inhibit channels composedof other Cxs (e.g., Cx37). L2 peptide is identical to the L2 domain on thecytoplasmic loop (CL; amino acids 119–144). Gap19 is a nonapeptidelocated within the L2 domain (amino acids 128–136). L2 and Gap19peptides block Cx43 hemichannels without blocking gap junctions andwithout blocking Cx40 hemichannels or Panx1 channels (other Cxs stillneed to be tested). CT10 and CT9 are the last 10 and last 9 amino acids ofthe C-terminal end (CT; amino acids 373–382 and 374–382, respectively).CT9/CT10 peptides remove the closure of Cx43 hemichannels with highmicromolar cytoplasmic Ca2+ concentration and thus stimulate theopening of hemichannels. This particular effect of the CT-peptides isindependent of the last amino acid (isoleucine 382) that is involved in linkingCx43 to scaffolding proteins involving ZO-1 interactions (the amino acidsequence 374–381 has the same effect as CT9/CT10; De Bock et al., 2012).

These domains, known as Gap26 and Gap27, are present onthe first and second extracellular loops, respectively. Accordingly,Gap26 and Gap27 peptides were indeed found to inhibit gapjunctional coupling (Evans and Boitano, 2001). Interestingly, sub-sequent work demonstrated that Gap26 and Gap27 peptides alsoinhibited unapposed hemichannels with which these peptides aresupposed to interact (Braet et al., 2003; Evans et al., 2006, 2012;Desplantez et al., 2012; Wang et al., 2012). Currently, clear evi-dence that these peptides indeed interact with the extracellularloops is only available for Gap26 (Liu et al., 2006). Neverthe-less, both Gap26 and Gap27 rapidly inhibit hemichannels withinminutes, followed by a somewhat delayed inhibition of gap junc-tion channels, often in the range of hours, with some exceptions(Matchkov et al., 2006). The exact mechanism of hemichannelblock is currently not known. It is known that Gap26/27 influencevoltage-dependent gating (Wang et al., 2012) but this does notexplain their inhibitory action on hemichannels opened by trig-gers other than voltage. It has been suggested that these peptidesinhibit hemichannels by just blocking the pore because of sterichindrance effects (Wang et al., 2007). However, this has been care-fully checked and it was found that this only occurs at very high(1 mM and above) concentrations (Wang et al., 2012). AlthoughGap26/27 peptides proved to be interesting tools to inhibit Cxhemichannels, their delayed inhibition of gap junction channelsremains a serious drawback. Moreover, Gap26/27 peptides alsoshow rather limited specificity toward different Cx types. Indeed,the extracellular loop domains mimic sequences that show pro-nounced homology between different Cx proteins. Thus, Gap27directed against Cx43 in astrocytes is also known to inhibit chan-nels or hemichannels composed of Cx37, a vascular Cx present inbrain blood vessels (Martin et al., 2005).

Besides peptides, antibodies have also been used to target thefree extracellular loops of unapposed Cx hemichannels result-ing in their inhibition. These tools offer additional advantagebecause they allow both functional blocking of the hemichan-nels and visualizing their distribution (Hofer and Dermietzel,1998; Clair et al., 2008; Riquelme et al., 2013). However, currentlysingle channel electrophysiological data showing hemichannelblock by an antibody directed against an extracellular epitope isonly demonstrated for Cx43 hemichannel activity induced in β-amyloid-treated astrocytes (Orellana et al., 2011c). Additionally,the specificity toward effects on other Cxs or Panxs still needs tobe determined.

To improve specificity toward different Cxs, peptides have beendevised that should target sequences on the intracellular domainsof the Cx protein. The intracellular sequences are poorly con-served and very different between different Cxs. The L2 peptideis identical to a sequence on the cytoplasmic loop of Cx43. Thispeptide is known to prevent the closure of gap junction channelsby binding to the CT tail, thereby preventing interaction of theCT tail with the cytoplasmic loop of Cx43 where the L2 domainis located (Seki et al., 2004; Hirst-Jensen et al., 2007 and referencestherein). Conceptually, interaction between the CT tail and the L2domain is thought to block the gap junction channel pore uponexposure to acidification according to a particle-receptor model,and exogenous addition of L2 peptide prevents this interactionand its associated channel closure. Unexpectedly, it was found




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that the L2 peptide inhibited Cx43 hemichannels, based on ATPrelease studies, indicating that this peptide has distinct effects onunapposed hemichannels and those incorporated into gap junc-tions (Ponsaerts et al., 2010). Further work with Gap19, a mimeticnonapeptide located within the Cx43 L2 domain, demonstrateda similar action; it inhibited single channel hemichannel currentsand ATP release via Cx43 hemichannels while it did not influ-ence gap junctional coupling after 30 min and stimulated couplingafter 24 h (Wang et al., 2013). Moreover, the effect appeared tobe specific for Cx43 as Gap19 had no effect on Cx40 hemichan-nels or Panx1 channels. While the effects of L2 and Gap19 areunderstood at a molecular operational scale, the full details ofwhy preventing interactions between the CT tail and the cytoplas-mic loop have such different effects on hemichannels as comparedto gap junction channels still needs further clarification. How-ever, distinct effects on hemichannels and gap junction channelshave been reported for other Cx channel influencing moleculesor conditions; for example, basic FGF (bFGF) and cytokines likeTNF-α or IL-1β have all been demonstrated to promote Cx43hemichannel function while inhibiting gap junctions (De Vuystet al., 2007; Retamal et al., 2007a). Given the specific actions ofL2 and Gap19 peptides at the level of both Cx types and channeltypes (hemichannels versus gap junction channels), these pep-tides offer promising possibilities to explore the role of astroglialCx43 hemichannels in the complex environment of intact neu-ral tissues, ex vivo or in vivo. Recent work has, for example,demonstrated that injection of L2 peptide in the rat amygdalastrongly suppresses fear memory (Stehberg et al., 2012; discussedfurther below under Section “Impact of Hemichannel-MediatedGliotransmission on Synaptic Activity and Behavior”). Equallyinteresting with respect to identifying the role and functions ofCx43 hemichannels is the G138R mutation of Cx43 in whicha Gly at position 138 is replaced by an Arg. This is one ofthe several mutants found in the inherited human disease ocu-lodentodigital dysplasia (ODDD). Importantly, G138R mutantCx43 is characterized by a loss-of-function of gap junctional cou-pling and a gain-of-function of hemichannels (Dobrowolski et al.,2007, 2008). Thus, this particular mutant Cx43 can be used tostimulate Cx43 hemichannel function as a complementary exper-imental approach to hemichannel inhibition. Recently, Torres et al.(2012) have exploited such an approach to investigate the role ofhemichannels in glial–neuronal communication.

ROLES OF GLIAL HEMICHANNELS IN HEALTH AND DISEASEASTROGLIAL Ca2+ WAVESInitially, gap junction channels were demonstrated to constitutethe pathway allowing the propagation of intercellular Ca2+ signal-ing between astrocytes, termed as Ca2+ waves (Finkbeiner, 1992;Venance et al., 1995). Then, after the discovery of an external com-ponent that also contributes to this interglial signaling process(Hassinger et al., 1996), the involvement of Cx43 hemichannelsand ATP release was identified (Cotrina et al., 1998; Stout et al.,2002). There is a consensus on the interpretation of these two setsof in vitro studies and it is admitted that both Cx channel func-tions are contributing. Indeed, they likely participate in variousproportions to Ca2+ waves depending on the brain structure, themode of stimulation to trigger the waves, the age and the in vitro

conditions (Scemes and Giaume, 2006; Leybaert and Sanderson,2012). In addition, several of these points could also account forthe alternative proposal claiming that in astrocytes the externalcomponent is not supported by Cx43 hemichannels but by Panx1channels (Suadicani et al., 2006; Scemes et al., 2007). This pointof controversy remains to be clarified in the future although it islikely that both might participate in various proportions underdifferent conditions.

More recently, several reports have confirmed that Ca2+ wavesin astrocytes are present in vivo in the normal brain (Hooglandet al., 2009; Kuga et al., 2011; Mathiesen et al., 2013) as well asin pathological situations (Kuchibhotla et al., 2009; Mathiesenet al., 2013). As expected, the occurrence, amplitude and extentof in vivo Ca2+ waves are much more limited compared to theinitial in vitro observations. However, as Ca2+ signaling is con-sidered as the mode of cellular excitability in astrocytes, suchwaves certainly play a role in the spatial and temporal featuresof neuroglial interactions. As a consequence, this means that inmany cases dynamic neuroglial interactions are not limited to thesole tri-partite synapse but also occur at a more integrated levelinvolving larger cerebral areas (Giaume et al., 2010). This situa-tion is also likely true at the gliovascular interface where thereis a high level of Cx expression between astroglial perivascu-lar endfeet (Yamamoto et al., 1990; Simard et al., 2003; Rouachet al., 2008) and where Ca2+ waves propagate from one initialendfoot to its neighbors (Mulligan and MacVicar, 2004). Suchfeatures might optimize the contribution of astrocytes to the con-trol of the cerebral blood flow. However, while a role of Cx43gap junction channels/hemichannels in pial arteriole dilation hasbeen demonstrated in vivo for the glia limitans in response to sci-atic stimulation (Xu and Pelligrino, 2007), a similar mechanismremains to be investigated for perivascular astrocytes within thebrain parenchyma.

HEMICHANNELS AS Ca2+ CHANNELSAs indicated above, glial cells express several Cxs and out of theseat least four of them (Cx26, Cx30, Cx32, and Cx43) have beendemonstrated to form functional hemichannels (Li et al., 1996;Rhee et al., 1996; Valiunas and Weingart, 2000; Ripps et al., 2004),and at least Cx26, Cx32, and Cx43 serve as a membrane path-way for cell (Sánchez et al., 2009, 2010; Schalper et al., 2010) orliposome (Schalper et al., 2010; Fiori et al., 2012) Ca2+ inflow. Ofcourse, brief opening of Cx hemichannels could serve to tran-siently increase the intracellular Ca2+ concentration that couldaffect a broad variety of physiological cell functions. For example,Cx43 hemichannels contribute to maintain higher intracellularlevels of free Ca2+ in cells treated with FGF-1 that proliferateand remain healthy (Schalper et al., 2008b). However, sustainedhemichannel opening could promote deleterious results, includ-ing cell death (Decrock et al., 2009, 2011), but the bell-shapedaction of intracellular Ca2+ might prevent this ending process.Panx1 has also been detected in glial cells, as mentioned above(Iglesias et al., 2009; Domercq et al., 2010; Orellana et al., 2012b).This glycoprotein has been shown to form channels in the ER per-meable to Ca2+ (Vanden Abeele et al., 2006; D’Hondt et al., 2011)but the permeability to Ca2+ of Panx1 channels present in the cellsurface remains elusive. Moreover, direct demonstration of either




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Cx- or Panx-based channels as a Ca2+ membrane pathway in anyglial cell type remains to be demonstrated.

ALTERNATIVE PATHWAY FOR GLUCOSE ENTRY IN INFLAMMATORYASTROCYTESThrough the so-called astrocyte–neuron lactate shuttle, astrocytesparticipate in an activity-dependent manner to the uptake of glu-cose from the blood supply and the delivery of lactate to neurons(Pellerin and Magistretti, 2012). In normal conditions, glucose andlactate transporters are the membrane elements that support thisastroglial contribution to brain metabolism. While glucose is wellknown to be essential for correct brain function, its homeostasis isaltered by inflammatory conditions (Allaman et al., 2011). In vitro,an inflammatory situation for astrocytes can be mimicked eitherby co-culturing them with microglia selectively activated by theendotoxin LPS or by directly treating them with pro-inflammatorycytokines, such as TNF-α and IL-1β. Interestingly, in both casesthese treatments induce an opposite regulation of Cx43 channelsat least in cultured astrocytes: a decrease in gap junctional commu-nication and an activation of hemichannels (Retamal et al., 2007a).In these conditions Cx43 hemichannels were shown to allowthe influx of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a glucose fluorescent analog, demon-strating that reactive or inflamed astrocytes can take up moreglucose via Cx43 hemichannels when their metabolic couplingthrough gap junction channels is reduced (Rouach et al., 2008).Such opening of Cx43 hemichannels may represent an alternativemetabolic pathway for glucose entry in astrocytes during patholog-ical conditions associated with an inflammation status (Sofroniewand Vinters, 2010). Interestingly, this observation identifies a path-way that could contribute to the increase of glucose uptake foundin several uncoupling situations (Tabernero et al., 2006); this wascorrelated with the up-regulation of GLUT-1 (glucose transporter1) expression and to the induction of the expression of GLUT-3,an isoform that is not normally expressed in astrocytes, as well fortype I and type II hexokinases, respectively (Sánchez-Alvarez et al.,2004). However, the respective contribution of hemichannels andglucose transporters in inflammatory conditions remains to beestablished. Because the permeability of the blood–brain barrier isincreased during the inflammatory response (Schnell et al., 1999),more glucose coming from the circulation could become availableto the brain. Thus, in addition to transporters, the entry of glu-cose through Cx43 hemichannels could contribute to an increaseof lactate formation by astrocytes that are adapted for anaero-bic metabolism. Finally, the opposite regulation of Cx43 channels(Retamal et al., 2007a) may lead to a failure in intercellular glu-cose trafficking (Rouach et al., 2008) that could be compensatedfor by the increase in glucose influx through Cx43 hemichannelsin individual astrocytes. Moreover, although not demonstratedyet, Cx43 hemichannels could also support lactate efflux to feedneurons since Cx43 gap junction channels in astrocytes are per-meable to lactate (Tabernero et al., 1996; Rouach et al., 2008). Asa whole, Cx43 hemichannel activation should certainly contributeto modify the metabolic status of reactive astrocytes, a situationthat should be taken into account when considering the role ofastroglia in brain inflammation.

IMPACT OF HEMICHANNEL-MEDIATED GLIOTRANSMISSION ONSYNAPTIC ACTIVITY AND BEHAVIORBy analogy with the concept of a neurotransmitter, the term “glio-transmitter” has been proposed to name active molecules that arereleased by astrocytes and regulate neuronal activity as well assynaptic strength and plasticity (Haydon and Carmignoto, 2006).There are mainly three candidates that are proposed to play thisrole: glutamate, ATP, and D-serine. All of them can be releasedby a Ca2+-dependent vesicular release mechanism in astrocytes(Zorec et al., 2012). However, at least glutamate and ATP havebeen demonstrated to permeate Cx43 hemichannels in astrocytes(Ye et al., 2003; Kang et al., 2008; Orellana et al., 2011b; Iglesiasand Spray, 2012) as well as Panx1 channels in microglia (Orellanaet al., 2011c), thus Cx43 and Panx1 channels could also play a rolein gliotransmission. Recently, the activation of Cx43 hemichannelsin astrocytes was shown to impact synaptic activity and plasticity(Stehberg et al., 2012; Torres et al., 2012). The first evidence comesfrom hippocampal acute slices in which a decrease in extracellu-lar Ca2+ concentration is generated by the local photolysis of aphotolabile Ca2+ buffer. This opens Cx43 hemichannels in astro-cytes through which ATP is released and activates P2Y1 purinergicreceptors on a subset of inhibitory interneurons, initiating the gen-eration of spikes by interneurons that in turn enhances synapticinhibition at glutamatergic synapses (Torres et al., 2012). A similarneuroglial loop of interaction acting as a negative feedback mech-anism is expected to occur during excessive glutamatergic activitythat is associated with a decrease in extracellular Ca2+ concen-tration (Rusakov and Fine, 2003). The second set of evidencecomes from in vivo experiments in which Cx43 hemichannelswere inhibited (Stehberg et al., 2012). In this study, the rat baso-lateral amygdala was microinfused with the TAT-L2 peptide thatselectively inhibits Cx43 hemichannels assumed to be only presentin astrocytes. Such in vivo blockade of Cx43 hemichannels dur-ing memory consolidation induces amnesia for auditory fearconditioning, as assessed 24 h after training, without affectingshort-term memory, locomotion, or shock reactivity. Moreover,the amnesic effect is transitory, specific for memory consolida-tion. Learning capacity recovers after co-infusion of the Cx43hemichannel blocker and a mixture of gliotransmitters includingglutamate and ATP. These observations suggest that gliotransmis-sion mediated by Cx43 hemichannels is necessary for fear memoryconsolidation at the basolateral amygdala. Thus, the study of Ste-hberg et al. (2012) represents the first in vivo demonstration of aphysiological role for astroglial Cx43 hemichannels in brain cogni-tive function. Finally, an in vivo contribution of Panx1 channels tocognition has recently been addressed by using a Panx1 knock outmouse (Prochnow et al., 2012). This constitutive knock out mousewas found to have increased excitability and potently enhancedearly and persistent long-term potentiation (LTP) responses inthe CA1 hippocampal region with additionally impaired spatialmemory and object recognition memory. However, while theexpression of Panx1 is well documented in hippocampal neurons(MacVicar and Thompson, 2010), its detection at the membraneof astrocytes is still debated (Iglesias et al., 2009; but see Orellanaet al., 2010, 2011c). Consequently, it is premature to state that thesePanx1-dependent changes in hippocampal models of learning andmemory have an astroglial origin.




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NEURONAL DEATH AND BRAIN PATHOLOGIESWhile there are now a number of reports indicating that the expres-sion of glial Cxs changes in many brain pathologies and injuries(Giaume et al., 2010), this remains to be investigated in detail forPanxs. On this basis, the question of Cx channels contributingto neuronal death and brain diseases has been debated for manyyears. Indeed, opposite perspectives regarding the roles of glial Cxsin neuronal death have been discussed and reviewed (see Kirchhoffet al., 2001; Rouach et al., 2002; Perez Velazquez et al., 2003; Nakaseand Naus, 2004; Farahani et al., 2005; Giaume et al., 2007; Orellanaet al., 2009). In general, this duality was attributed to differencesin (i) the experimental models (in vitro, ex vivo, and in vivo) or theprotocols used to induce neuronal death, (ii) the mode (phar-macology versus transgenic animals) of Cx channel inhibitionselected, (iii) the type of neuronal death or pathology investigated,and (iv) the time points considered. However, retrospectively theopposite roles attributed to Cx channels could be simply due tothe fact that for a long time this question was solely taking intoaccount the gap junctional function of Cxs. Indeed, in most cases,interpretation of the data was not considering the hemichanneland channel functions of Cxs and Panxs, respectively. Neverthe-less, the development of compounds and transgenic animals thatdiscriminate between Cx gap junction channels, Cx hemichan-nels and Panx channels (see above) has allowed identification oftheir respective contribution in various in vitro and ex vivo mod-els of brain pathologies (see Kielian, 2008; Orellana et al., 2009;Bennett et al., 2012). Although hemichannels have been reportedto be involved in several brain pathologies or injuries (reviewedin Bennett et al., 2012) as well as infection (Karpuk et al., 2011;Xiong et al., 2012), we will illustrate this situation by two recentexamples that demonstrate the impact of glial Cx hemichanneland Panx channel activation in cells on neuronal death.

Modifications in the pattern of Cx and Panx expression in gliaare associated with phenotypic changes observed during neuroin-flammation and the “reactive gliosis” that is a common featureof most brain diseases (see Sofroniew and Vinters, 2010). Indeed,activated microglia and reactive astrocytes exhibit changes in theirrespective pattern of expression for Panx1 and Cx43 as well as intheir hemichannel function leading to neuronal damage and death(Bennett et al., 2012; Orellana et al., 2012b). When an inflam-matory context is mimicked in vitro by using LPS treatment,Cx43 hemichannel activity is triggered in astrocytes through theproduction of TNF-α and IL-1β by activated microglia. Theseevents are not observed when astrocytes are cultured from Cx43knock out mice (Retamal et al., 2007a). Furthermore, in neuron–astrocyte co-cultures the N-methyl-D-aspartate (NMDA)-inducedneuronal excitotoxicity is reinforced by TNF-α + IL-1β treat-ment, while such a potentiating effect is not observed in culturesof neurons alone treated with the two cytokines (Froger et al.,2010). This observation suggests that the well-known protectiverole of astrocytes against excitotoxicity (Chen and Swanson, 2003)is impaired under pro-inflammatory conditions when astroglialCx43 hemichannels are activated. Neuron–astrocyte co-cultures,made with astrocytes from Cx43 knock out and neurons fromwild-type mice, revealed that the NMDA excitotoxicity is notreinforced by the pro-inflammatory treatment applied on theseco-cultures made with Cx43-deficient astrocytes (Froger et al.,

2010). This result suggests that astroglial Cx43 is involved inthe potentiated neurotoxicity induced by pro-inflammatory treat-ments. Interestingly, application (<30 min) of mimetic peptides(Gap26, Gap27) that inhibit Cx43 hemichannels and have no effecton Cx43 gap junctional communication (Retamal et al., 2007a),prevents the potentiated neurotoxicity response induced by pro-inflammatory cytokines (Froger et al., 2010; Orellana et al., 2012b).

Connexin hemichannels might also play a role in the patholog-ical context of neurodegenerative diseases. Indeed, it is becomingincreasingly clear that glial cells are critical players in the progres-sive neurodegeneration of Alzheimer’s disease (Kraft et al., 2013).Among others, the amyloid cascade represents one of the keypathways involved in the pathogenesis of the disease (see Pim-plikar, 2009). Using separate primary cultures and co-cultures ofmicroglia, astrocytes, and neurons it was shown that treatmentwith the β-amyloid peptide (Aβ) first activates microglia, resultingin the opening of Cx43 hemichannels and Panx1 channels in thesecells. Such channel activations allow the release of glutamate andATP that impair neuronal survival. Aβ-activated microglia alsoproduces TNF-α and IL-1β that, as indicated above, induce Cx43astroglial hemichannel activation that again results in the release ofglutamate and ATP (Orellana et al., 2011c). In fine these gliotrans-mitters originating from glial cells trigger neuronal damage andinduce their death. Thus, such a cascade of Cx glial hemichanneland Panx channel activation could partially account for the pro-gression of the neurodegenerative disease. These in vitro and exvivo observations could be related to the changes in Cx43 expres-sion observed in reactive astrocytes that contact amyloid depositin a murine model of Alzheimer’s disease (Mei et al., 2010) and inbrain from Alzheimer’s patients (Nagy et al., 1996; Koulakoff et al.,2012).

CONCLUDING REMARKSDuring the last decade our view and understanding of the structure(Maeda and Tsukihara, 2011), mutations (Pfenniger et al., 2011),properties (Rackauskas et al., 2010), and biological roles (Kar et al.,2012) of Cx channels has been incredibly enlarged, and to someextent this is also true for Panx channels (MacVicar and Thomp-son, 2010; Penuela et al., 2013). Interestingly, in glial cells thisfeature is exemplary because in addition to their well-documentedmolecular support for their two channel functions, non-channelfunctions have been also identified for Cxs, including cell adhesionmechanisms involved in radial migration of cortical neurons (Eliaset al., 2007; Cina et al., 2009), modulation of P2Y purinergic recep-tors signaling (Scemes, 2008), regulation of cytoskeletal dynamics(Olk et al., 2010) and control of gene expression (Iacobas et al.,2004). Such a view could even be further enlarged by the recentreport of another new class of membrane protein called calciumhomeostasis modulator CALHM1 that shares structural similari-ties with Cx, Panx, and innexin channels and functional propertiessimilar to hemichannels, such as ATP release in taste transduction(Siebert et al., 2013; Taruno et al., 2013).

In the present review, we have focused on a Cx/Panx-basedchannel function which 10 years ago was only sparsely consideredin the central nervous system and in neuroglial interactions. Sincethen, the identification of external and intracellular signals thattrigger the activation of hemichannels, the understanding of their




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biophysics and their regulation have provided a better understand-ing of the roles that glial Cxs and Panxs play in healthy and diseasedbrains. Indeed, as reported above, there is now strong evidence tostate that Cx and Panx channels in glia have an impact on neu-ronal activity and survival. However, there are at least two aimsthat remain to be achieved in order to determine whether and howthe contribution of glial Cxs and/or Panxs is causal or secondaryin normal and pathological situations. One is to distinguish whatis due to gap junction channels versus hemichannels, the otheris to discriminate between Cx hemichannels and Panx channels.To achieve these goals it is essential to have available pharmaco-logical and genetic tools with glial cell type, Cx type, and Panxtype specificities (see Giaume and Theis, 2010). Through a simplepharmacological approach this is unlikely to be achieved sinceCx and Panx channels are sensitive to the same agents so fardescribed. However, strategies using short-term treatment withmimetic peptides (Evans et al., 2012) or antibodies targeting extra-cellular domains (Riquelme et al., 2013) could be developed for allthe glial Cxs/Panxs and their specificity demonstrated. In con-trast, due to the cytoplasmic location of their two channel ends,gap junction channels are likely less easy to target by a pharmaco-logical strategy. Another more accurate approach consists in thedevelopment of transgenic mice with appropriate mutations thatprevent channel pairing without affecting hemichannel activity orthat block one but not the other channel function. Alternatively,small interfering RNA (siRNA) and transgenic/viral techniqueshave been used with some success (see Giaume and Theis, 2010).But here again, the expression of multiple Cxs in glial cells makesthe task difficult with genetic and molecular approaches. A recentreport by Nedergaard’s group of an enhanced synaptic plastic-ity (LTP) due to the engraftment of human glial progenitors into

immunodeficient mice (Han et al., 2013) gives weight to achievesuch difficult challenges. Indeed, upon maturation in these graftedmice, human astrocytes were shown to be coupled to host astro-cytes, suggesting a contribution of Cx channels in neuroglialinteractions during synaptic plasticity. In line with such contribu-tion, double Cx43/Cx30 knock out is associated with reduced LTP(Pannasch et al., 2011). Also, the observation of an up-regulationof astroglial Cxs in reactive astrocytes contacting amyloid plaquesin a transgenic murine model of Alzheimer’s disease has been vali-dated in human patient brain samples (Nagy et al., 1996; Koulakoffet al., 2012), giving another example of the value in searching forstrategies that block Cx channels.

ACKNOWLEDGMENTSThis review arose from discussions made possible by a Collo-quium Abroad grant from the Peter Wall Institute for AdvancedStudies, University of British Columbia, in partnership with theCollège de France and the Fondation Hugo du Collège de France,Paris, France, as well as funds from the Vice President Research &International, University of British Columbia. This study includesreference to research that was supported by Grants from ECOS-CONICYT (to Christian Giaume and Juan C. Sáez), The Caisse deRetraite et de Prévoyance des Clercs et Employés de Notaires andthe Ligue Européenne Contre la Maladie d’Alzheimer (to Chris-tian Giaume), the Fund for Scientific Research Flanders (to LucLeybaert) and Belgian Science Policy (Interuniversity AttractionPoles to Luc Leybaert), Fondecyt project 1120214 (to Juan C.Sáez), 1111033 (to Juan C. Sáez), Chilean Science MillenniumInstitute (P09-022-F; to Juan C. Sáez), the Canadian Institutes forHealth Research (to Christian C. Naus), and the Heart and StrokeFoundation of Canada (Christian C. Naus).

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Conflict of Interest Statement: Theauthors declare that the research wasconducted in the absence of any com-mercial or financial relationships thatcould be construed as a potential con-flict of interest.

Received: 23 April 2013; paper pendingpublished: 21 May 2013; accepted: 21June 2013; published online: 17 July 2013.Citation: Giaume C, Leybaert L, NausCC and Sáez JC (2013) Connexin andpannexin hemichannels in brain glialcells: properties, pharmacology, and roles.Front. Pharmacol. 4:88. doi: 10.3389/fphar.2013.00088This article was submitted to Frontiersin Pharmacology of Ion Channels andChannelopathies, a specialty of Frontiersin Pharmacology.Copyright © 2013 Giaume, Leybaert,Naus and Sáez. This is an open-accessarticle distributed under the terms of theCreative Commons Attribution License,which permits use, distribution andreproduction in other forums, providedthe original authors and source are cred-ited and subject to any copyright noticesconcerning any third-party graphics etc.


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